Gel Electrophoresis

What is the agarose made from?

an extract of red algae

What are the two different types of DNA testing?

1. Y chromosome testing. 2. Mitochondrial testing

What are the five major components of an electrophoresis apparatus?

1. electrical current 2. the test sample (DNA, etc) 3. gelatin medium (agarose) that the sample moves through 4. a liquid to conduct the electrical current (usually a buffer) 5. a stain used to highlight the migrated samples

What are three errors that could affect the outcome of any gel electrophoresis procedure?

1. placing dyes in the wrong wells. 2. piercing the well with the micropipette. 3. running the gel for too long.

What are the key steps to the electrophoresis procedure?

1. test sample preparation- isolating and purifying the samples as well as the addition of the "tracking" dye. 2. gel preparation- several types of gel materials can be used. Agarose is commonly used. When agarose solidifies it forms a matrix. 3. gel loading- gel is placedd into the electrophoresis chamber and a running buffer is added to the chamber covering the entire gel. 4. Sample tracking- the run continues until the tracking dye reaches the end of the gel. Power is turned off and gel is removed. 5. gel staining- a variety of staining techniques can be used to enhance the visability of the DNA bands left at various locations along the gel. Once stained, the gel can be refrigerated and stored. Often the gels are photographed before disposal to provide a permanent data record.

Who invented gel electrophoresis?

Oliver Smithies

What is agarose gel electrophoresis used for?

analysis of nucleic acids and proteins. separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. Used to determine presence and size of PCR products. anode (+) cathode (-). small DNA runs faster than larger DNA. Gel electrophoresis separated DNA according to size.

Define the process of gel electrophoresis

applying an electrical current to a gelatin-like substance containing biological samples. When mixtures are placed within the wells of the gel and an electrical current is applied , the molecules travel through the gel and separate from one another according to each molecule's charge, size and shape.

What are other techniques used to separate and identify molecules?

distillation, crystallization, liquid chromatography, gas chromatography, atomic absorption

Why don't all dyes travel the same distance or the same direction in the wells?

each dye has a different molecular weight, which will cause them to travel different distances. The dyes will travel different directions depending on their charges. Negatives will travel towards the positive end of the gel and the positives will travel towards the negative end of the gel.

What is agarose?

it acts as a type of molecular strainer and prevents all the molecules from moving too quickly. The electric current moves DNA molecules across the agarose. The agarose gel is used to visualize the fragments. It can be used to separate DNA molecules ranging from several hundred nucleotides in length to ober 10,000 nucleotides.

Define Gel Electrophoresis

laboratory method used to separate mixtures of DNA according to molecular size. Molecules are separated by being pushed through an electrical field through a gel that contains small pores.

What is mitochondrial testing?

mitochondria are inherited solely from the mother. A mother's mitochondrial DNA can be compared with that of her children and those of her daughter's children, etc. through the maternal line.

What is the function of the electric current?

shocks it so that it will move towards the positive and negative sides.

Why is the fact that DNA has a negative charge so important in the gel electrophoresis process?

so the negatively charged DNA can move toward the positive pole of the electrophoresis chamber.

What is the purpose of ethidium bromide?

stains the DNA. Ethidium bromide binds tightly to the DNA double helix and glows when illuminated with ultraviolet light. This lets researchers see where separated DNA fragments end up.

What is Y chromosome testing?

the Y chromosome is passed on from the father to son. The Y chromosome is the ideal chromosome for determining if one male is directly related to another man. A grandfather, father and son all share the same STR pattern on the Y chromosome.

What is the function of electrophoresis buffer?

the buffer keeps the pH within a narrow range. The charge is also transmitted via ions provided by the buffer.

Define restriction enzymes

the enzymes which break DNA molecules at internal positions are called restriction endonucleases. Enzymes that degrade DNA by digesting the molecule from the ends of the DNA strand are termed endonucleases.

Why is it important to match more than one STR to determine relatedness?

the more STR sequences that are analyzed, the more significant th match that can be made in DNA forensics. In related people, the number of times an STR repeats in one area on a single chromosome is likely to be the same.

Why does DNA have an overall negative charge?

the phosphate groups in the DNA backbone carry negatively charged oxygens giving a DNA molecule an overall negative charge.

What is the function of the wells in the gel?

the sample being ran in gel electrophoresis is loaded into the wells of the gel.

Archaeologists would like to determine which mummies are related to King Tut. Which DNA fingerprinting technique would be the most useful to determine the male relatives? Why?

they would use Y chromosome testing. Because they will be able to tell who the male relatives are through the Y chromosome which is passed from father to son.

What causes pore size in agarose?

varies by using different concentrations of agarose. The higher concentration of agarose, the smaller the pore size.

Briefly summarize how gel electrophoresis is used to separate molecules

when the molecules are placed in the individual wells they are all at the same distance because all the wells were created with the same comb. Gel electrophoresis is applying an electrical current to separate the molecules. Once gel electrophoresis is ran, the negative dyes will run towards the positive end of the gel and the positive dyes will run towards the negative end of the gel. The heavier dyes will run a shorter distance compared to the lighter dyes, this is how they are separated out.